Over 600 different carotenoids have been described from carotenogenic organisms found among bacteria, yeast, fungi and plants. Currently only two of them, .beta.-carotene and astaxanthin are commercially produced in microorganisms and used in the food and feed industry. .beta.-carotene is obtained from algae and astaxanthin is produced in Pfaffia strains which have been generated by classical mutation. However, fermentation in Pfaffia has the disadvantage of long fermentation cycles and recovery from algae is cumbersome. Therefore, it is desiderable to develop production systems which have better industrial applicability, e.g., can be manipulated for increased titers and/or reduced fermentation times.
Two such systems using the biosynthetic genes form Erwinia herbicola and Erwinia uredovora have already been described in WO 91/13078 and EP 393 690, respectively. Furthermore, three .beta.-carotene ketolase genes (.beta.-carotene .beta.-4-oxygenase) of the marine bacteria Agrobacterium aurantiacum and Alcaligenes strain PC-1 (crtW) [Misawa, 1995, Biochem. Biophys. Res. Com. 209, 867-876] [Misawa, 1995, J. Bacteriology 177, 6575-6584] and from the green algae Haematococcus pluvialis (bkt) [Lotan, 1995, FEBS Letters 364, 125-128] [Kajiwara, 1995, Plant Mol. Biol. 29, 343-352] have been cloned. E. coli carrying either the carotenogenic genes (crtE, crtB, crtY and crtI) of E. herbicola [Hundle, 1994, MGG 245, 406-416] or of E. uredovora and complemented with the crtW gene of A. aurantiacum [Misawa, 1995] or the bkt gene of H. pluvialis [Lotan, 1995][Kajiwara, 1995] resulted in the accumulation of canthaxanthin (.beta.,.beta.-carotene-4,4'-dione), originating from the conversion of .beta.-carotene, via the intermediate echinenone (.beta.,.beta.-carotene-4-one).
Introduction of the above mentioned genes (crtW or bkt) into E. coli cells harbouring besides the carotenoid biosynthesis genes mentioned above also the crtZ gene of E. uredovora [Kajiwara, 1995][Misawa, 1995], resulted in both cases in the accumulation of astaxanthin (3,3'-dihydroxy-.beta.,.beta.-carotene-4,4'-dione). The results obtained with the bkt gene are in contrast to the observation made by others [Lotan, 1995], who using the same experimental set-up, but introducing the H. pluvialis bkt gene in a zeaxanthin (.beta.,.beta.-carotene-3,3'-diol) synthesising E. coli host harbouring the carotenoid biosynthesis genes of E. herbicola, a close relative of the above mentioned E. uredovora strain, did not observe astaxanthin production.
However, functionally active combinations of the carotenoid biosynthesising genes of the present invention with the known crtW genes have not been shown so far and even more importantly there is a continuing need in even more optimized fermentation systems for industrial application.